Conventionally, for nucleic acid sequence determination, gene mutation detection, and the like, a technique has been used in which: nucleic acid molecules are amplified in droplets of a “water-in-oil type” emulsion; the amplification product is fixed on the surface of beads; the beads are collected by breaking the droplets of the emulsion; and then the amplification product is analyzed.
For example, U.S. Patent application Publication No. 2012/183967 discloses a method for detecting gene mutation by the BEAMing method. In the detection method according to U.S. Patent application Publication No. 2012/183967, the steps of binding oligonucleotides to beads (step 1), preparing a microemulsion (step 2), PCR cycling (step 3), magnetic capture of beads (step 4), sequence differentiation (step 5), and flow cytometry (step 6) are performed, and then, mutant DNA molecules are counted.
In the preparation of the microemulsion (step 2), an aqueous phase that contains magnetic beads and a target gene, and an oil phase that contains a reagent for preparing an emulsion are mixed together and stirred, whereby the microemulsion is prepared. With the technique according to U.S. Patent application Publication No. 2012/183967, 200 microliters of the aqueous phase is added by a dropping method to 400 microliters of the oil phase, whereby a water-in-oil type microemulsion is prepared. The addition of the aqueous phase by the dropping method is performed for about one minute while the mixture is being stirred with a stirrer at 1400 RPM. Then, the mixture is further stirred for 30 minutes.
In the magnetic capture of beads (step 4), in order to break the microemulsion, an NX buffer is added to the microemulsion. After the solution is stirred, the oil phase is separated from the aqueous phase by centrifugation, and the top oil phase is removed. To the resultant aqueous phase, the NX buffer is added, and the oil phase is removed by centrifugation once again. Then, the magnetic beads are attracted by a magnet, and the supernatant is carefully removed using a pipette. Further, the magnetic beads are washed with a PCR buffer three times using magnetic separation, and finally re-suspended in the PCR buffer. In this manner, in step 4, in order to break the microemulsion, operations of removal of the oil phase, magnetic separation, and the like are performed a plurality of times.